Tuberculosis has re-emerged as a global health concern and the World Health Organization estimates that Mycobacterium tuberculosis infects one third of the world population. The ability to survive and multiply within the host macrophage by arresting phagosome maturation is a hallmark of M. tuberculosis pathogenicity. However, when bacteria are unable to arrest phagosome maturation, such as in activated macrophages, they are delivered to the lysosome and killed. Solubilized lysosomes isolated from primary macrophages possess potent antimycobacterial properties that are associated with ubiquitin and ubiquitin-derived peptides. Ubiquitin-derived peptides obtained from digestion of full-length ubiquitin with lysosomal proteinases or the synthetic peptide Ub2 were mycobactericidal in vitro. Further study of the synthetic antimycobacterial peptide Ub2 is necessary to determine the target and specificity of these molecules. It is hypothesized that there are mycobacterial factors involved in susceptibility and resistance to host-derived antimicrobial peptides and these factors contribute to M. tuberculosis virulence. This project will elucidate the mode of action of ubiquitin-derived peptides through identification and characterization of mycobacterial hyper-susceptible and hyper-resistant mutants and will define the role of host antimicrobial peptides in M. tuberculosis infection. In Specific Aim 1, we will identify mycobacterial factors involved in susceptibility and resistance to ubiquitin-derived antimicrobial peptides. Mycobacterium mutants that are hyper-susceptible and hyper-resistant to the antimycobacterial action of the ubiquitin-derived peptide Ub2 have been isolated. The genetic locus disrupted in these mutants will be identified and classified functionally. Mutants predicted to encode efflux pumps, ABC transport systems, and membrane proteins will be prioritized for subsequent experiments. In Specific Aim 2, we will determine contribution to M. tuberculosis virulence of mycobacterial factors involved in susceptibility and resistance to ubiquitin-derived antimicrobial peptides. Resting, activated, and autophagic macrophages will be infected with M. tuberculosis antimicrobial resistance mutants to determine if they exhibit altered phenotypes. Mouse infection studies with M. tuberculosis antimicrobial resistance mutants will determine the contribution of these factors toward virulence.